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Genetic studies of beak and feather disease virus

Bassami, Mohammad Reza (2000) Genetic studies of beak and feather disease virus. PhD thesis, Murdoch University.

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The aim of the studies reported in this thesis was to genetically characterise the genome of beak and feather disease virus (BFDV), a circovirus causing psittacine beak and feather disease (PBFD). The objective was to utilise the sequence data to develop additional genetic-based tools for the detection of the virus and facilitate further investigation of the pathogenesis and epidemiology of the disease.

To determine an optimal method of preparing purified virus DNA for cloning and sequencing, several methods for the extraction and purification of BFDV from tissues of PBFD-affected birds were compared. Maximum recovery of virus from tissues was achieved using a combination of solubilisation of virus with the chlorinated hydrocarbon Arklone™, differential centrifugation of the virus through a 45% sucrose cushion, followed by isopycnic ultracentrifugation of the virus in caesium chloride gradients.

The circular, single-stranded DNA of one isolate of BFDV from a sulphur crested cockatoo with PBFD was cloned and sequenced. The genome of this strain was 1993 nucleotides in length. Analysis of the assembled double-stranded replicative form of the virus DNA demonstrated 7 open reading frames (ORF), 3 in the virion strand and 4 in the complementary strand, potentially encoding 7 viral proteins greater than 8.7 kDa. Fligh amino acid sequence identity was demonstrated between a potential 33.3 kDa protein product of ORF1 of BFDV and the replicase-associated protein (Rep) of porcine circovirus (PCV), and nanoviruses (plant circoviruses). No significant similarity was found between BFDV and chicken anaemia virus (CAV), another member of the family Circoviridae. A potential stem-loop structure and a nonanucleotide motif (TAGTATTAC) within it, similar to that found in PCV, nanoviruses and geminiviruses, was demonstrated in the virion strand of the BFDV genome. Comparison of the deduced amino acid sequences of ORF2 of BFDV and PCV demonstrated 29.1 % identity, and in both viruses ORF2 was located on the complementary strand, beginning close to or within the hairpin loop. The findings provided further evidence of that BFDV has a close relationship with PCV and the plant circoviruses but not with CAV.

To determine the extent of genetic variability within BFDV, the complete nucleotide (nt) sequence of an additional 8 isolates and partial nucleotide sequence of one other isolate recovered from various psittacine species across Australia was determined. All isolates had the same basic structure including the position of the ORFs, the presence of hairpin or stem-loop structure between ORF1 and ORF2, the nonanucleotide motif (TAGTATTAC) therein, the 3 motifs of the Rep protein involved in rolling circle replication, and the P-loop motif. The size of the genome in the isolates ranged from 1992 to 2018 nt and overall nt identity of the isolates compared to the first strain that was sequenced ranged from 84% to 97%; the variation was due to a combination of point mutations and a number of deletions and insertions ranging from 1 to 17 nt in size detected in both the coding regions and non-coding regions. Phylogenetic analysis grouped the isolates into 4 clusters but there were no apparent regional differences or differences related to the psittacine species from which they were derived. While 7 ORFs with the potential to encode proteins greater than 8.7 kDa were detected in the original strain, only 3 of these ORFs were detected in all 10 BFDV isolates for which sequence data was obtained: ORF1, ORF2 and ORFS. The ORF1 and ORF2 probably encoded the Rep and capsid proteins, respectively, but the protein encoded by ORFS, if any, has to be determined.

A universal PCR assay was designed that consistently detected BFDV in a range of psittacine species affected with PBFD from a variety of geographic regions within Australia. A Southern blot technique was also developed to detect BFDV genomic DNA in PBMC of infected birds, and also confirm the specificity of PCR products generated from feathers or peripheral blood mononuclear cells (PBMC) of PBFDaffected birds.

A set of degenerate primers was designed to try and amplify genomic material of circoviruses related to BFDV and PCV and suspected to be present in other animal species. With these primers, PCR amplification of a region of the genome of a novel circovirus present in Senegal doves affected with a PBFD-like syndrome was achieved.

Item Type: Thesis (PhD)
Murdoch Affiliation: Division of Veterinary and Biomedical Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Wilcox, Graham and Raidal, Shane
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